We have discovered a novel inhibitor of neutrophil extracellular trap (NET) formation that circulates in the umbilical cord blood of newborn infants (Neonatal NET-inhibitory Factor; nNIF). NETs are extracellular lattices of decondensed chromatin extruded by polymorphonuclear leukocytes (PMN) and decorated with histones and antimicrobial proteins to protect against infection by trapping and killing microbes. Yet, NET formation, if dysregulated, can cause inflammatory tissue damage leading to significant morbidity and mortality in pathologic inflammatory syndromes. This project aims to determine whether NETs protect or harm in models of neonatal sepsis, a syndrome all too common in neonates which leads to significant morbidity and mortality. We have demonstrated impaired NET formation by PMNs isolated from neonates. This led to studies demonstrating that nNIF circulates in the developing human fetus but decreases or disappears rapidly after birth. We have also identified two additional NET-Inhibitory Peptides (NIPs) that also inhibit NET formation by neonatal and adult human PMNs in a potent, concentration-dependent manner. Further data now show that NIPs inhibit NET formation in vivo in murine models of peritonitis and sepsis. Finally, NIP treatment improves survival in preclinical models of infection and inflammation suggesting that NIPs may decrease inflammatory tissue damage and mortality in sepsis. Based on these published and unpublished observations, this proposal outlines three specific aims that will provide novel insights into nNIF, other NIPs, and NET inhibition in neonatal sepsis. We will elucidate the regulatory mechanisms governing nNIF expression in utero and after birth. We will determine the mechanism(s) of action for NIPs. We will also determine whether nNIF alters in vivo NET formation, inflammatory tissue damage, and survival in preclinical models of neonatal sepsis, and compare these outcomes to adult sepsis models. These studies will significantly advance the fields of neonatal medicine and neutrophil biology by determining how NIPs inhibit NET formation by PMNs and by determining whether NET formation contributes to inflammatory tissue damage and survival in models of neonatal sepsis. Our laboratory is uniquely poised to determine the NET-inhibitory mechanism(s) of NIPs and to explore the benefits and potential harm of inhibiting NET formation in sepsis and, ultimately, other inflammatory disorders of newborns, children, and adults. These studies are translational and innovative: They examine a novel neonatal modulatory factor, nNIF, and represent the first use of specific endogenous inhibitors of NET formation to determine whether NETs protect or harm in neonatal experimental sepsis. The impact of these studies will, however, extend beyond neonatal sepsis to other inflammatory syndromes of prematurely born infants, such as necrotizing enterocolitis, and to other inflammatory conditions of older children and adults.